The stilbenes isolated from Photorhabdus species bacteria are known to have antibiotic activity (Paul et al. Journal of Chemical Ecology, 7: 589-597 (1981), and Hu et al., Canadian Journal of Microbiology. 44: 1072-1077 (1998). However, these compounds have not been shown to have biological activity other than anti-microbial and nematicidal activities.
A similar compound, resveratrol, has been disclosed as having cancer preventive (Jang, et al. 1997, Science, 275, 218, U.S. Pat. No. 6,008,260) and protein kinase C inhibitory activities (Garcia-Garcia et. al., 1999). There are also many stilbene derivatives that are well-known in the art to have a wide range of activities and are widely distributed in nature. There is a growing interest in stilbene derivatives because of a range of activities that have been observed in some of the naturally occurring as well as some of the synthetic stilbenes.
It is known in the art that substitution on the various position on the two phenyl rings of the basic stilbene structure results in a great diversity of compounds, including those with one or two substituents on one or both of the phenyl rings of the stilbene structure (Shudo K., 1988, U.S. Pat. No. 4,723,028; Hensley, K. L., et al., WO99/59561, Kunihiro N., 1983, JP58159410; Genji I., 1995, JP07053359 and GB1465661) and those with three or more substituents on the phenyl rings (Koichi, S. et al., 1986, EP0170105; Shozo Y., et al., 1986, JP08337523; and Charpentier B. et al., 1992, WO92/19583). Compounds with other substitution on the phenyl ring, such as derivatives of vitamin A (Ney, U. M., et al. 1987, Dermatologica, 175:93-99) and vitamin D (WO 00/26167) are well-known in the art. Several publications (WO92/16486, WO99/40056, WO01/95859 and Cushman M. et. al. (1992, J. Med. Chem., 35:2293-2306) disclosed compounds that are derived from 3,4,5-trimethoxyl stilbene, and these compounds also showed anti-neoplastic activity and modest activity of modulating cytokines (WO01/95859).
Fang J. M. et al. (1988, Phytochem., 27(5): 1395-1397) also described a stilbene oxide that has two methoxyl groups at 3 and 5 positions, however, there is no substituent in between, and no specific applications are described for the treatment of inflammatory and/or autoimmune diseases. Syah Y. M. et al., described a new stilbene, andalasin A (2000, Fitoterapia., 71: 630-635) that shares some similarity to the compounds of the current invention. However it is a complicated natural product, and it is only demonstrated to have weak antinematodal and moderate antifungal properties. Dudek S. P. et al (2001, J. Am. Chem. Soc., 123: 8033-8038) described ferrocene-containing stilbene derivatives that also have two methoxyl groups at 3 and 5 positions, as well as a substituent in between. However, these compounds contain metal for completely different applications, and have different structural properties that are unrelated to the compounds of the current invention. Treadwell E. M. et al. described schweinfurthins that are also stilbene derivatives (2002, Org. Lett., 4: 3639-3642). These stilbenes share the structural nature of the current invention, however the substituents between the two hydroxyl groups are all related to the geranyl group, and they have only demonstrated anticancer activity.
Recently, when working on stilbene derivatives, the inventors discovered a group of stilbenes with a unique substitution pattern of two hydroxyl groups, or their derivatives, in position 3 and 5 and a substituent in between and their unexpected activities in mediating T-cell, cytokines, growth factors and inflammatory mediators. The present invention is related to these known and novel stilbene compounds, their synthesis, their unexpected activity, pharmaceutical compositions and their use for treatment of disorders associated with these activities such as many inflammatory and/or autoimmune diseases.
Inflammatory diseases, whether of a chronic or acute nature, represent a substantial problem in the healthcare industry. Chronic inflammation is considered to be inflammation of a prolonged duration (weeks or months) in which active inflammation, tissue destruction and attempts at healing are proceeding simultaneously (Robbins Pathological Basis of Disease by R. S. Cotran, V. Kumar, and S. L. Robbins, W. B, Saunders Co., p. 75, 1989). Although chronic inflammation can follow an acute inflammatory episode, it can also begin as an insidious process that progresses with time, for example, as a result of a persistent infection (e.g., tuberculosis, syphilis, fungal infection) which causes a delayed hypersensitivity reaction, prolonged exposure to endogenous (e.g., elevated plasma lipids) or exogenous (e.g., silica, asbestos, cigarette tar, surgical sutures) toxins, or, autoimmune reactions against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, psoriasis, inflammatory bowel disease, eczema). Chronic inflammatory diseases therefore, include many common medical conditions such as rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis, and chronic inflammatory lung and airway diseases (e.g., asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis).
Psoriasis is a common, chronic inflammatory skin disease, characterized by rapid multiplication and turnover of the epithelial cells with a consequent thickening of the epidermis, as well as inflamed swollen skin lesions covered with silvery white scaling patches and raised, inflamed, thickened and scaly lesions, which itch, burn, sting and bleed easily. It is therefore a disease characterized not only by inflammation, but also by proliferation of cells. In respect of proliferation, therefore, it has similarities to cancers, and both psoriasis and cancers can be described as proliferative diseases. In approximately 10% of patients, psoriasis is accompanied by pronounced arthropathic symptoms that are similar to the changes seen in rheumatoid arthritis. Approximately 2 to 3% of the U.S. population suffers from psoriasis, with 250,000 new cases being diagnosed each year. The compounds of the invention possess specific activity against psoriasis.
Eczema is a chronic inflammatory skin disorder, also known by terms used to describe the disorder as atopic dermatitis, neurodermatitis, disseminated lichen simplex chronicus, or atopic eczema. Atopic eczema affects 10 to 20 percent of children in Western populations. Eczema is characterized by physiologic, immuno pathologic, and pharmacological abnormalities that involve the skin. These abnormalities include: 1) a lowered threshold to itch stimuli, 2) a hypersensitivity to α-adrenergic agonists and to cholinergic agents, 3) a very dry hyperkeratotic skin which has decreased water-holding capacity, 4) a marked tendency to produce lichenification in response to friction and scratching, and 5) a tendency for the skin to be colonized with bacteria.
Inflammatory bowel disease (IBD) comprises ulcerative colitis and Crohn's disease, both of which exhibit common clinical features including chronic, relapsing inflammation of the gastrointestinal tract, abdominal pain, abdominal mass, persistent diarrhea, blood loss, fever, malnutrition, and fatigue. The prognosis in IBD is unpredictable; patients may relapse several times per year or may not relapse for several years. In addition, there are many systemic complications that accompany this disease with the most common being arthritis. Symptoms of arthritis occur in one fourth of all people with IBD. Joint inflammation occurs most often when the colon is involved in the disease process and flares when the bowel disease is most active. This form of inflammatory arthritis does not cause permanent deformity and is often short lived. Other complications of this disease include eye inflammation (iritis, conjunctivitis and episcleritis), mouth inflammation (mucositis), skin inflammation (erythema nodosum and pyoderma gangrenosum), musculoskeletal abnormalities (ankylosing spondylitis), renal complications (kidney stones and fistulas to urinary tract), gallstones and other diseases of the liver (e.g. hepatitis) and biliary system (sclerosing cholangitis). Unfortunately, in many cases, long-term disease (>10 years) can lead to more severe complications such as colonic cancer and extraintestinal carcinomas. The precise etiology of these diseases remains unclear. However, it is now recognize that IBD results from dysregulation of the immune system with many facets resembling other auto-immune diseases that involve cytokines such as IFN-γ and TNF-α. Currently, approximately 2 million people in the United States suffer from IBD with males and females affected equally.
Protein kinase is implicated in many diseases, including cancers, as well as being a factor in diabetes, atheromatous plaque and epidermal proliferation (including psoriasis). The compounds of the invention possess specific protein kinase inhibiting activity. It is believed that protein kinase inhibitors may be of great importance in the control of uncontrolled cellular reproduction, i.e. in cellular reproduction disorders. For example DNA-PK is a serine/threonine protein kinase that is composed of a very large catalytic polypeptide and a DNA binding/targeting regulatory subunit (Ku autoantigen). Ku was first recognized as a heterodimeric (p70/p80) nuclear phosphoprotein that reacted with sera from patients suffering from autoimmune diseases lupus erythematosus and scleroderma polymyositis. Casein kinase II (Ck2) is a serine/threonine kinase that phosphorylates acidic proteins such as casein. Ck2 has been shown to play multiple roles inside the cell and can be activated by numerous growth factors, hormones and cytokines. Ck2 has multiple substrate targets inside the cell which are ultimately involved in the regulation of DNA, RNA and protein synthesis. Ck2 plays a role in controlling mitogenic signalling and neuritogenesis. Ck2 has been shown to be involved with numerous disease states. Elevated Ck2 levels have been demonstrated in solid human tumors and rapid proliferating non-neoplastic tissue such as colorectal mucosa. Ck2 activity was much higher in metastatic melanoma and in cells transformed by human cytomegalovirus. Infection of animals with protoxoan parasite resulted in fatal lymphoproliferative syndrome that is associated with the over expression of Ck2. Ck2 activity has also been demonstrated to be elevated in Alzheimer's disease.
Amson et al. (1989. Proc. Nat. Acad. Sci. 86: 8857-8861) showed that the 33-kD product of the PIM gene is highly expressed in the liver and spleen during fetal hematopoiesis. In contrast, it is only slightly expressed in circulating granulocytes in adults. It was overexpressed in hematopoietic malignancies, particularly in myeloid and lymphoid acute leukemias. The results implied a physiologic role of the Piml oncogene during hematopoietic development and a deregulation of the gene in various leukemias. Saris et al. (1991. EMBO J. 10: 655-664) provided evidence that both the murine and the human Piml gene products are protein-serine/threonine kinases. In the mouse, at any rate, they showed that the gene encodes both a 44- and a 34-kD protein, the former being an amino-terminal extension of the latter which is synthesized by alternative translation initiation at an upstream CUG codon. Ark et al. (1991. Genomics 10: 385-389) provided refined mapping of the Pim-1 locus in the mouse and used the Pim-1 gene as a marker for further genetic analysis of t-haplotypes on mouse chromosome 17. To understand the function of Pim-1 and its role in hematopoietic development, Laird et al. (1993. Nucleic Acids Res. 21: 4750-4755) generated mice deficient in Pim-1 function. Pim-1-deficient mice were ostensibly normal, healthy, and fertile; however, detailed analysis demonstrated a correlation of Pim-1 deficiency with erythrocyte microcytosis, whereas overexpression of Pim-1 in transgenic mice resulted in erythrocyte macrocytosis.
Recent studies on the molecular basis or neoplastic transformation have identified a family of genes, designated oncogenes, whose aberrant expression causes tumorigenesis. For example, the RNA tumour viruses possess such an oncogene sequence whose expression determines neoplastic conversion of infected cells. The tyrosine kinase Lck is expressed primarily in different types of hematopoietic cells. The Lck protein is found in thymocytes and mature T cells and has been reported to be expressed in mature mouse splenic B cells. Campbell et al. (1992. Mol. Cell. Biol., 12: 2315). Lck inhibitors are of value in the treatment of a number of such disorders (for example, the treatment of autoimmune diseases), as Lck inhibition blocks T cell activation. The treatment of T cell mediated diseases, including inhibition of T cell activation and proliferation, is a particularly preferred embodiment of the present invention.
Accordingly, a specific inhibitor of these kinases can be useful in investigating the mechanism of cancerogenesis, cell proliferation, differentiations and autoimmunology and it can be effective in prevention and chemotherapy of cancer and other pathological proliferative conditions. Hence the compounds according to the present invention can be useful in the treatment of pathological proliferation and autoimmune disorders in mammals, including humans. A human or animal, e.g. a mammal, can thus be treated by a method comprising the administration thereto of a therapeutically effective amount of one of the compounds of the invention. Amelioration of the disease state or disorder from which the human or animal is suffering can be achieved. Typical examples of such disorders are benign and malignant tumours, including leukaemia such as myeloblastic leukaemia, lymphoma, sarcoma, neuroblastoma, Wilm's tumour, malignant neoplasm of the bladder, breast, lung or thyroid and neoplasias of epithelial origin, such as mammacarcinoma. Moreover, they can be useful in the treatment of epidermal hyper-proliferation, such as psoriasis. The compounds of the invention can also be useful in inhibiting the development of atheromatous plaque and restenosis, in the control of angiogenesis, as anti-metastatic agents and in treating diabetic complications. They have also utility in the control of immune system diseases, e.g. as immuno-suppressants.
Cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor alpha (TNF-α) play major roles in inflammation. Overproduction of TNF-α and IFN-γ has been linked to several inflammatory diseases including psoriasis, eczema, inflammatory bowel disease (IBD) and rheumatoid arthritis. In psoriasis, the skin plaques of patients with psoriasis contain large numbers of activated T-cells as compared with normal skin. These T-cells release factors, including the cytokines which promote activation, hyper-proliferation and altered differentiation of keratinocytes thus forming the plaque. Anti-TNF drugs have been used in the treatment of rheumatoid arthritis for some time and recently, Remicade, an anti-TNF-α drug has been approved to treat inflammatory bowel diseases (IBD).
IFN-γ is produced by CD8+ T cells, a sub-group of CD4+ (Th1 type) T cells, and macrophages. This factor may be present at high levels in tissues afflicted by autoimmune processes. IFN-γ promotes a number of pro-inflammatory aspects of immune responses including the up-regulation of major histocompatibility complex (MHC) and adhesion molecule expression, cytokine (TNF-α) formation and the release of chemical mediators (e.g. nitric oxide). For a number of autoimmune diseases, the disease-associated inflammatory process is associated with an increased availability of IFN. IFN-γ increases endothelial cell adhesion molecule expression and thereby can influence leukocyte recruitment to inflammatory sites. Lupus-prone mice treated with anti-IFN-γ antibody were protected from disease development. The genetic knockout of the IFN-γ receptor prevented autoantibody production and glomerulonephritis in a lupus-prone mouse strain. Moreover, administration of IFN-γ to mice in a SLE disease model intensified disease parameters while mice given anti-IFN-γ antibody exhibited increased remission and survival. IFN-γ increased disease severity in mouse MS disease models. Neutralization of IFN-γ with antibodies or administration of IFN-γ ameliorates symptoms in various animal autoimmune models, effects that are often directly related to the stage of disease when these agents are provided. Thus, IFN-γ may have a strong impact on autoimmune disease progression or resolution, actions that may be specific for the particular condition. However, in lupus models, the neutralization of IFN-γ has a beneficial effect regardless of the time of introduction of the antibody. IFN-γ is an effective target for the treatment of psoriasis, eczema, multiple sclerosis, and IBD.
Vascular endothelial growth factor (VEGF) is a cytokine that is involved in a variety of physiological events, including induction of vascular hyperpermeability or edema. It plays a key role in the pathogenesis of many inflammatory disorders such as pulmonary hypertension and inflammatory airway diseases. VEGF is normally overexpressed in hyperproliferative diseases such as psoriasis and the levels of VEGF are substantially elevated in psoriasis. VEGF is also a key regulator in physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF also has been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions.
Leukotriene B4 (LTB4) is an important mediator of inflammation derived from the arachidonic acid pathway. It promotes adherence and chemotaxis of white blood cell's and degranulation and, consequently, plays an important role in the inflammatory process. LTB4 has been reported to be involved in lipopolysaccharide-induced sepsis and endotoxemia.
In a particular embodiment, the compounds of the present invention are useful for the treatment of the aforementioned exemplary disorders irrespective of their etiology, for example, for the treatment of transplant rejection, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, lupus, graft vs host disease, T-cell mediated hypersensitivity disease, psoriasis, eczema, Hashimoto's thyroiditis, Guillain-Barre syndrome, cancer, contact dermatitis, allergic disease such as allergic rhinitis, asthma, ischemic or reperfusion injury, or atopic dermatitis. Transplant rejection (graft v. host disease) and surgical adhesions are also affected by protein kinases. The protein kinase inhibition and anti-inflammatory properties of the compounds of this invention give rise to utility in surgery to reduce transplant rejection and surgical adhesions.